1. Field of the Invention
This invention relates generally to sensing the presence of magnetic particles, and more particularly to quantitatively measuring accumulations of such particles by means of AC magnetic excitation and inductive sensing of the amplitude of the resulting oscillations of the magnetic moments of the particles at the excitation frequency.
2. Discussion of Prior Art
Much attention has been given to techniques for determining the presence, and possibly the level of concentration, of minute particles in a larger mixture or solution in which the particles reside. It is desirable in certain circumstances to measure very low concentrations of certain organic compounds. In medicine, for example, it is very useful to determine the concentration of a given kind of molecule, usually in solution, which either exists naturally in physiological fluids (for example, blood or urine) or which has been introduced into the living system (for example, drugs or contaminants).
One broad approach used to detect the presence of a particular compound of interest, referred to as the analyte, is the immunoassay, in which detection of a given molecular species, referred to generally as the ligand, is accomplished through the use of a second molecular species, often called the antiligand, or the receptor, which specifically binds to the first compound of interest. The presence of the ligand of interest is detected by measuring, or inferring, either directly or indirectly, the extent of binding of ligand to antiligand.
A discussion of several detection and measurement methods appears in U.S. Pat. No. 4,537,861 (Elings et al.). That patent discloses several ways to accomplish homogenous immunoassays in a solution of a binding reaction between a ligand and an antiligand, which are typically an antigen and an antibody. Elings discloses creation of a spatial pattern formed by a spatial array of separate regions of antiligand material and ligand material dispersed to interact with the spatial array of separate regions of antiligand material for producing a binding reaction between the ligand and the antiligand in the spatial patterns and with the bound complexes labeled with a particular physical characteristic. After the labeled bound complexes have been accumulated in the spatial patterns, the equipment is scanned to provide the desired immunoassay. The scanner may be based on fluorescence, optical density, light scattering, color and reflectance, among others.
The labeled bound complexes are accumulated on specially prepared surface segments according to Elings, or within an optically transparent conduit or container by applying localized magnetic fields to the solution where the bound complexes incorporate magnetic carrier particles. The magnetic particles have a size range of 0.01 to 50 microns. Once the bound complexes are accumulated magnetically within the solution, the scanning techniques previously described are employed.
Magnetic particles made from magnetite and inert matrix material have long been used in the field of biochemistry. They range in size from a few nanometers up to a few microns in diameter and may contain from 15% to 100% magnetite. They are often described as superparamagnetic particles or, in the larger size range, as beads. The usual methodology is to coat the surface of the particles with some biologically active material that causes them to bond strongly with specific microscopic objects or particles of interest (e.g., proteins, viruses, cells, DNA fragments). The particles then become xe2x80x9chandlesxe2x80x9d by which the objects can be moved or immobilized using a magnetic gradient, usually provided by a strong permanent magnet. Thus, the Elings patent is an example of tagging using magnetic particles. Specially constructed fixtures using rare-earth magnets and iron pole pieces are commercially available for this purpose.
Although these magnetic particles have only been used in practice for moving or immobilizing the bound objects, some experimental work has been done on using the particles as tags for detecting the presence of the bound object. This tagging is usually done by radioactive, fluorescent, or phosphorescent molecules which are bound to the objects of interest. A magnetic tag, if detectable in sufficiently small amounts, would be very attractive because the other tagging techniques all have various important weaknesses. For example, radioactive methods present health and disposal problems. The methods are also relatively slow. Fluorescent or phosphorescent techniques are limited in their quantitative accuracy and dynamic range because emitted photons may be absorbed by other materials in the sample. See Japanese Patent Publication 63-90765, published Apr. 21, 1988 (Fujiwara et al.).
Because the signal from a very tiny volume of magnetic particles is exceedingly small, it has been natural that researchers have tried building detectors based on Superconducting Quantum Interference Devices (xe2x80x9cSQUIDxe2x80x9ds). SQUID amplifiers are well known to be the most sensitive detectors of magnetic fields in many situations. There are several substantial difficulties with this approach, however. Since the pickup loops of the SQUID must be maintained at cryogenic temperatures, the sample must be cooled to obtain a very close coupling to these loops. This procedure makes the measurements unacceptably tedious. The general complexity of SQUIDs and cryogenic components renders them generally unsuitable for use in an inexpensive desktop instrument. Even a design based on so-called xe2x80x9chigh Tcxe2x80x9d superconductors would not completely overcome these objections, and would introduce several new difficulties. See Fujiwara et al.
There have been more traditional approaches to detecting and quantifying the magnetic particles. These have involved some form of force magnetometry in which the sample is placed in a strong magnetic gradient and the resulting force on the sample is measured, typically by monitoring the apparent weight change of the sample as the gradient is changed. An example of this technique is shown in U.S. Pat. Nos. 5,445,970 and 5,445,971 to Rohr. A more sophisticated technique measures the effect of the particle on the deflection or vibration of a micromachined cantilever. See Baselt et al., A Biosensor based on Force Microscope Technology, Naval Research Lab., J. Vac Science Tec. B., Vol 14, No. 2 (pg. 5) (April 1996). These approaches are all limited in that they rely on converting an intrinsically magnetic effect into a mechanical response. This response must then be distinguished from a large assortment of other mechanical effects such as vibration, viscosity, and buoyancy.
There would be important applications for an inexpensive, room-temperature, desktop instrument which could directly sense and quantify very small amounts of magnetic particles.
Broadly speaking, the present invention provides a method and an apparatus for directly sensing and measuring very small accumulations of magnetically susceptible particles, e.g., magnetite, and consequently, their coupled substances of interest.
The magnetic particles or beads are coupled by known methods to analyte particles, thereby providing magnetic sample elements or magnetic bound complexes. A well-defined pattern of the magnetic sample elements is deposited on a surface on a holder. The surface may be flat. A high-amplitude, high-frequency magnetic field is then applied to excite the particles in the sample. The field causes the particles to behave as a localized dipole oscillating at the excitation frequency. The fields from the sample are closely coupled to a sensor, such as an array of inductive sensing coils, which may be fabricated in a gradiometer configuration. This configuration makes the sensing coils mostly insensitive to the large, uniform field that is used to excite the sample. Moreover, the geometry of the coils is designed to match the spatial pattern of the sample so as to provide a large response that varies distinctively with the relative positions of the sample and coils.
The voltage induced across the sensor is carefully amplified and processed by phase-sensitive detection. An inductive pickup from the drive field itself may serve as the reference signal to the phase detector circuit. The output of the phase detector is further filtered and digitized.
The signal amplitude is modulated by moving the sample with respect to the sensor. This allows the rejection of signals due solely to imbalance of the sensor, non-uniformity of the drive field, cross-talk in the circuitry, or any other source of apparent signal which is not due to the sample itself. The digitized shape of the signal amplitude with respect to the sample position is compared to the theoretical response shape using appropriate curve-fitting techniques, providing a very accurate estimate of the magnetic content of the sample in the face of inherent instrument noise and drift.